Attenuated pseudorabies virus which has a deletion in at least a portion of a repeat sequence and vaccine containing same

ABSTRACT

Attenuated pseudorabies viruses are provided which comprise DNA including a sequence essential for replication of the attenuated virus, at least a portion of which is present in a sequence essential for replication of a naturally-occurring pseudorabies virus, from which at least a portion of a repeat sequence has been deleted. These viruses are useful as vaccines for immunizing animals against pseudorabies virus disease. 
     The invention also provides methods of preparing attenuated pseudorabies viruses.

BACKGROUND OF THE INVENTION

Within this application several publications are referenced by arabicnumerals within parentheses. Full citations for these references may befound at the end of the specification immediately preceding the claims.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

The ability to isolate viral DNA and to clone this DNA into bacterialplasmids has greatly expanded the approaches that can be used to makeviral vaccines. The approach in this application is to modify the viralDNA sequence while in the cloned state in a plasmid, modifications whichinclude, but are not limited to, insertion, deletion, or single ormultiple base change. The modified DNA is then inserted back into theviral genome for the purpose of rendering the virus nonpathogenic. Theresulting live virus product can be used to elicit an immune responseand be protective as a vaccine, or in any other situation wherein anonpathogenic virus infection of an animal is required.

One group of animal viruses, the herpesviruses or herpetoviridae, is anexample of a class of viruses amenable to this approach. These virusescontain 100,000 to 150,000 base pairs of DNA as their genetic material,and several areas of the genome have been identified that aredispensible for the replication of virus in vitro in cell culture.Modification of these regions of the DNA are known to lower thepathogenicity of (to attenuate) the virus for the animal species. Forexample, inactivation of the thymidine kinase gene renders human herpessimplex virus non-pathogenic (1), and pseudorabies virus of swinenon-pathogenic (2) and (3).

Removal of part of the repeat region renders human herpes simplex virusnon-pathogenic (4) and (5). A repeat region has been identified inMarek's disease virus that is associated with viral oncogenicity andchanges in this region correlates with lack of oncogenicity (6). Thesemodifications in the repeat region do not teach the construction ofattenuated pseudorabies viruses with deletions in repeat sequences. Adifferent region in herpesvirus saimiri has similarly been correlatedwith oncogenicity (7). A region in pseudorabies virus has been shown tobe deleted in naturally occurring vaccine strains (8). This deletion ispartly responsible for lack of pathogenicity, however it does not occurin a repeat sequence and does not suggest attenuation resulting from adeletion in a repeat sequence.

The general conclusion from the literature is that herpesviruses containnonessential regions of DNA in various parts of the genome, and thatmodification of these regions can lead to attenuation of the virus andderivation of a vaccine or non-pathogenic strain. The degree ofattenuation of the virus is important in the utility of the virus as avaccine. Deletions which cause too much attenuation of the virus willresult in a vaccine that fails to elicit an adequate immune response.

The herpesviruses are known to cause a variety of latent and recurrentinfections in human and other vertebrates and are even known to infect afungus and an oyster. Among the conditions associated with herpesvirusinfection are fever blisters caused by herpes simplex type 1, genitalherpes caused by herpes simplex type 2, and chickenpox in children andshingles in adults caused by herpes zoster infection. A Class D herpesvirus known as pseudorabies virus (PRV) induces Aujeszky's disease, anacute and often fatal nervous condition, in domestic and wild animals.

The natural host of pseudorabies virus is swine, in which infection iscommonly inapparent but may be characterized by fever, convulsions andparalysis. Pseudorabies virus also infects cattle, sheep, dogs, cats,foxes and mink, where infection usually results in death of the host.The predominant visible feature of pseudorabies virus infection isintense pruritis generally resulting in host mutilation of the involvedarea. Violent excitement, fits and paralysis, all symptoms ofencephalomyelitis, precede death which usually occurs within a few daysfollowing onset.

The pseudorabies virus genome has been mapped (See FIG. 1). The genomeis known to include, in order, a unique long region, an internalinverted repeat sequence, a unique short region, and a terminal invertedrepeat sequence.

Pseudorabies virus disease in swine is of serious concern bygovernmental bodies worldwide. In the U.S., infected swine cannot besold except to slaughterhouses. Several individual states haveseparately enacted eradication control practices against pseudorabies.The R & D trend among traditional vaccine manufacturers has been toemphasize research leading to products that are based upon the subunitvaccine approach rather than using live viruses. This departure fromlive virus vaccines is due mainly to the recognized safety aspect ofsubunit vaccines, and their unlikelihood of containing infectious liveviruses. It is well known that traditional live virus vaccines canrevert to virulence which would seem to maintain a source of the diseaseduring an eradication program. Attenuated pseudorabies viruses whichhave a deletion in at least a portion of a repeat sequence are adeparture from most current live virus vaccines, and from the directionin which the vaccine industry as a whole is moving. These viruses sufferdeletions in the repeat region and in other regions which cannot berepaired, i.e. the viruses cannot revert to virulence and are safe. Avaccine comprised of these viruses combines the safety aspect of subunitvaccines with the commercial and efficacious advantages of live virusvaccines.

Vaccines against pseudorabies virus are available. These vaccines aregenerally produced by the technique of cell culture attenuation. It isknown that some live virus vaccines produced by this method containdeletions in the unique short region of the viral genome (8), howeverthese viruses do not have deletions in repeat sequences.

On May 15, 1985 European Patent Publication No. 0 141 458 was published.This European Patent Publication is based upon European PatentApplication No. 84201474.8, filed on October 12, 1984, and is entitled"Deletion Mutant of a Herpesvirus and Vaccine Containing Same".

This publication discloses the construction of attenuated pseudorabiesviruses which have sequences of DNA deleted from the genome. Among thetypes of deletions are deletions in the unique short region anddeletions in the repeat sequences. However this application does notprovide enough detail to teach attenuating deletions in repeat sequencesnor does it suggest the unexpected attenuating nature of deletions inrepeat sequences. Specifically the inventors state that a small deletionin the repeat unit at the position of the Hind III site is not ofimportance for virulence in pigs, thereby indicating directly that theywere not aware of the importance of the repeat region in attenuation. Inany event, this application was published in a foreign country less thanone year before the filing date of this application.

It is also known that deletions in the thymidine kinase (Tk) gene of thepseudorabies virus, located in the unique long region of the genome,renders the virus non-pathogenic but reduces the immune response in thehost animal as well. U.S. Pat. No. 4,514,497, entitled "Modified LivePseudorabies Viruses", discloses temperature resistant pseudorabiesviruses which have deletions in the TK gene only and does not teach orsuggest pseudorabies viruses which are attenuated by deleting portionsof repeat sequences. In addition our attenuated pseudorabies viruseshave never been selected for any kind of temperature resistance.

An ideal live virus vaccine is non-pathogenic and produces a strongimmune response. It is therefore desirable to produce a livepseudorabies virus vaccine which would be non-pathogenic and produce astrong immune response in host animals.

This invention concerns attenuated pseudorabies viruses, i.e.pseudorabies viruses with lowered pathogenicity, which are useful as avaccine to elicit a strong immune response or in other situationswherein a nonpathogenic viral infection of an animal is required. Theseviruses have a deletion in at least a portion of a repeat sequence. Whenused to immunize animals against pseudorabies virus disease, theseviruses provide the advantages of not killing foreign animals or beingpassed to companion animals. Additionally, the viruses are stable, i.e.they do not revert to a pathogenic strain, and they provide a goodimmune response.

SUMMARY OF THE INVENTION

Attenuated pseudorabies viruses are provided which are comprised of DNAincluding a sequence essential for replication of the attenuated virusfrom which at least a portion of a repeat sequence has been deleted. Atleast a portion of the sequence essential for replication of theattenuated virus is present in a sequence essential for replication of anaturally-occurring pseudorabies virus. Vaccines comprised of aneffective immunizing amount of a virus of this invention and a suitablecarrier are useful for immunizing animals against pseudorabies virusdisease.

The invention also provides methods of preparing attenuated pseudorabiesviruses and methods of preparing pseudorabies virus vaccines from theseviruses.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a pseudorabies virus genome map including a BamHI restrictionmap showing the location of the PstI #7 fragment. UL=Unique longsequence; US=Unique short sequence; IR=Internal repeat sequence;TR=Terminal repeat sequence.

FIG. 2 is a pseudorabies virus BamHI #5 restriction map showing thelocation of the repeat deletion in S-PRV-001.

FIG. 3 (A) is a pseudorabies virus BamHI #5 restriction map showing thelocation of the repeat deletion in S-PRV-002 (B) is a pseudorabies virusPstI #7 restriction map showing the location of the deletion in thethymidine kinase gene region.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns attenuated pseudorabies viruses. Theseviruses are comprised of DNA which includes a sequence essential forreplication of the attenuated virus from which at least a portion of arepeat sequence has been deleted. At least a portion of this sequence ispresent in a sequence essential for replication of a naturally-occurringpseudorabies virus. The viruses are not temperature resistant, i.e. theyhave not been selected for replication at a temperature higher thantemperatures which are permissive for the wild type. A virus which isnot temperature resistant is defined herein as a virus capable ofreplicating only at temperatures permissive for the wild type.Additionally, the DNA sequence essential for replication of theattenuated virus may be derived from a naturally-occurring pseudorabiesvirus.

The deletion may be totally comprised of a portion of a repeat sequenceor a portion of a repeat sequence and a portion of an adjacent uniqueregion, i.e. the deletion may occur in a junction region. Additionally,a sequence nonessential for replication may be deleted from one or bothrepeats. Furthermore, at least a portion of the essential sequence ofone repeat or one entire repeat may be deleted.

The invention also concerns attenuated pseudorabies viruses which arecomprised of pseudorabies viral DNA from which at least a portion of arepeat sequence and a sequence not located within a repeat have beendeleted. In one embodiment of the invention a nonessential sequencepresent in both repeats and a sequence not located within a repeat havebeen deleted. In a preferred embodiment of the invention the deletedsequence not located within a repeat is at least a portion of a gene. Ina specific embodiment of the invention the deleted sequence not locatedwithin a repeat is at least a portion of the gene encoding pseudorabiesthymidine kinase. An attenuated pseudorabies virus with a deletion inboth repeats and a deletion in the gene encoding pseudorabies thymidinekinase has been constructed. This virus is designated S-PRV-002 and isdeposited under ATCC Accession No. VR 2107.

The invention also concerns an attenuated pseudorabies virus whichcomprises pseudorabies viral DNA from which at least a portion of arepeat sequence other than the junction region or the immediate earlygenes has been deleted. An attenuated pseudorabies virus with a deletionin both repeat sequences upstream of the promoter for the immediateearly genes has been constructed. This virus has been designatedS-PRV-00l and is deposited under ATCC Accession No. VR 2106.

The invention also concerns vaccines for pseudorabies virus disease.These vaccines are comprised of an effective immunizing amount of anattenuated pseudorabies virus having at least a portion of a repeatsequence deleted and a suitable carrier. Typically the suitable carrieris a physiologically balanced culture medium containing stabilizingagents, e.g. sugars, salts and proteins. In one embodiment of theinvention the vaccine for a swine contains an effective immunizingamount of an attenuated pseudorabies virus equal to about 10³ to about10⁹ plaque forming units (pfu) per dose, while the effective immunizingamount for a dog, cat, sheep or bovine animal, i.e. cattle, is fromabout 10⁴ to about 10⁶ pfu per dose.

The invention also concerns methods of immunizing animals againstpseudorabies virus disease by administering to the animals a suitabledose of a vaccine of this invention. The vaccine may be administered byintramuscular, subcutaneous, interperitoneal or intravenous injection.Additionally, the vaccine may be administered intranasally or orally.

The invention also concerns a method of preparing attenuatedpseudorabies viruses. This method involves isolating wild typepseudorabies viral DNA and using restriction enzyme digestion to produceDNA restriction fragments. These restriction fragments are purified byagarose gel electrophoresis to obtain specific DNA fragments which aretreated with appropriate enzymes, known to those skilled in the art, toproduce modified viral DNA fragments. These modified viral DNA fragmentsare capable of binding to bacterial plasmid DNA sequences. Suitablebacterial plasmids are separately treated with appropriate restrictionenzymes, known to those skilled in the art, to produce bacterial plasmidDNA sequences capable of binding to modified viral DNA fragments. Thesebacterial plasmid DNA sequences are then combined with the modifiedviral DNA fragments under suitable conditions to allow the viral DNA tobind to the bacterial DNA and form a viral-bacterial plasmid.

The viral-bacterial DNA plasmid is then mapped by restriction enzymes togenerate a restriction map of the viral DNA insert. The viral-bacterialDNA plasmid is then treated with restriction enzymes known in the art tocause at least one deletion in the viral DNA sequence of theviral-bacterial DNA plasmid. This plasmid, containing at least onedeletion in the viral DNA sequence, is transfected with wild type intactpseudorabies viruses into animal cells. The animal cells are maintainedunder suitable conditions to allow the wild type pseudorabies viral DNAto regenerate the wild type virus and a small percentage of viruseswhich have recombined with the viral DNA sequence of the plasmid. Someof these recombined viruses have deletions in their genome as a resultof deletions in the viral DNA insert of the plasmid. These viruses areidentified and subsequently plaque purified away from the wild typevirus.

In a preferred embodiment of the invention the viral DNA restrictionfragment inserted into the bacterial plasmid includes at least a portionof a repeat sequence. In a specific embodiment of the invention thedeletion in the viral DNA sequence of the viral-bacterial DNA plasmidleads to deletions in both repeat sequences of the recombined virus. Inanother preferred embodiment of the invention the viral DNA restrictionfragment inserted into the bacterial plasmid includes the gene encodingpseudorabies thymidine kinase. A specific embodiment of the inventioninvolves deletions in the viral DNA sequence of the viral-bacterial DNAplasmids which lead to deletions in both repeat sequences and the geneencoding pseudorabies thymidine kinase of the recombined virus.

The invention also concerns methods of preparing pseudorabies virusvaccines. These methods include cultivating the viruses in rollerbottles or a suspension of microcarrier beads, e.g. Sephadex® beads. Theviruses may also be cultivated by batch fermentation.

CONSTRUCTION OF ATTENUATED PSEUDORABIES VIRUSES Materials and Methods

All methods employed are those in standard usage in molecular biologyresearch laboratories or minor variations of those methods.

The starting wild-type pseudorabies virus for all these studies was theShope strain obtained from the USDA, which is a virulent virusrecommended for use in challenge studies. The virus was propagated lessthan 4 times in Vero cells for challenge stocks, and less than 10 timesfor the manipulation and purification of vaccine strains.

All enzymes employed are readily available from commercial sources andwere used according to the recommendations of the suppliers. Forexample, restriction enzymes were obtained principally from New EnglandBiolabs (Biolabs), Bethesda Research Laboratories (BRL), and IBICorporation, among others. Bal 31 nuclease was from BRL; DNA T4 ligasewas from Biolabs; calf-intestine phosphatase was fromBoehringer-Mannheim Corp.; and the Klenow fragment of E.coli DNApolymerase I was from Biolabs.

Plasmid DNA was prepared essentially by the methods described in (10).Viral DNA preparation is described in (11). DNA preparations from smallsamples of plasmid transformed cells ("Mini preps" ) were made accordingto the methods described in (10).

DNA fragments were purified from agarose gels following electrophoreticresolution according to (10). Phenol extraction was used for low meltingpoint agarose gels and electro-elution for regular agarose gels. DNA wastransformed into E.coli strain HB101 by methods described in (10). DNAwas transfected into animal cells essentially by he method of (12) and(13).

The structure and order of restriction fragments in DNA was analyzed byrestriction digestion, resolution of the fragments by agarose gelelectrophoresis and hybridization to defined radiolabeled DNA or RNAprobes following transfer of restriction fragments from the gel tofilters by a blot procedure ("Southern blot" analysis) described in(10). Viruses obtained from tissue culture monolayers infected withpseudorabies recombinant viruses were selected for the presence of thethymidine kinase genes in HAT medium, or the absence of the gene in Budrmedium in procedures described in (14). The presence of the thymidinekinase enzyme was assayed by the C¹⁴ thymidine labeling methodsdescribed in (15).

Restriction fragments of DNA were rendered blunt using the Klenowfragment of E.coli DNA polymerase as described in (10). Removal ofterminal phosphate residues by digestion with calf-intestine phosphataseis also as described in (10).

Construction of S-PRV-001

The manipulations that resulted in a pseudorabies virus with a deletionin the repeat region were as follows. The thymidine kinase region ofpseudorabies virus Shope strain was cloned as the 4300 bp PstI #7fragment (pSY344). Contained within this region is an approximate 3200bp BamHI fragment called Bam #11. The Bam #11 fragment has been reportedto contain the pseudorabies virus thymidine kinase gene by marker rescueexperiments and sequencing data (16). We have also shown by markerrescue experiments that our Bam #11 clone rescues a thymidine kinasepseudorabies mutant and thus contains the thymidine kinase gene.

The MluI site lies within the thymidine kinase structural gene (16).Plasmids containing this insert were cut, digested and religated tocreate deletions at the Mlul site. Plasmids were analyzed for the sizeof the deletion, and a plasmid with a deletion of approximately 300 bp(pSY513) was used in transfection in the presence of wild typepseudorabies virus DNA in animal cells. Viruses were selected in thepresence of the drug bromodeoxyuridine. Candidate recombinant viruseswere plaque purified away from wild type pseudorabies, a preparation ofDNA was made, and the DNA was analyzed by restriction digestion andSouthern blotting (10) to prove that a deletion was present in the viralgenome. From these results, the location of the deletion in BamHI #5 hasbeen determined and is shown in FIG. 2. By these methods a pseudorabiesvirus (S-PRV-001) was isolated which had suffered a deletion in bothcopies of BamHI #5 fragment and no deletion in Pst #7 fragment. Thevirus retains an intact and active thymidine kinase gene as determinedby l4C-thymidine incorporation assay.

Construction of S-)PRV-002

An attenuated pseudorabies virus containing multiple deletions in thegenome as a result of homologous recombination between cloned fragmentsof pseudorabies DNA and intact wild type pseudorabies DNA has beenconstructed. The methodology was as described in the construction ofS-PRV-001.

The plasmid clone used in the recombination was derived from the PstI #7cloned fragment of pseudorabies virus. It should be noted that while thecloned PstI #7 fragment maps to the long unique region, it is alsoweakly homologous to BamHI #5 repeat fragment. The PstI #7 fragment waspurified from a PstI digest of viral DNA following agarose gelelectrophoresis and was cloned into the Pst site of pBR 322 to formpSY344.

Deletions were made in this plasmid around the MluI site bY cuttingplasmid DNA with MluI restriction enzyme followed by Ba131 nucleasedigestion. The DNA was digested for varying lengths of time (10 to 60seconds), samples were analyzed by agarose gel electrophoresis todetermine the approximate extent of digestion. Samples digested between100 and 2000 Base pairs were religated to circularize the DNA andtransformed into E.coli. Plasmid pSY513 with a deletion of approximately300 base pairs was used for recombination with viral DNA. The plasmidDNA was mixed with intact pseudorabies virus DNA and the mixture wastransfected into rabbit skin cells. The expected location of deletionswould be in regions of the DNA homologous to the plasmid DNA; i.e., inPstI #7 (or BamHI #11 contained there) and in BamHI #5 which hybridizesto a limited extent with PstI #7 fragment. The viruses recovered fromtransfection were selected with the drug bromodeoxyuridine which wouldselect for recombinants that were defective in thymidine kinaseactivity. The thymidine kinase gene resides on fragment PstI #7 and adeletion in the gene would result from homologous recombination at thatsite. The viruses were plaque purified two times and examined forchanges in the DNA by restriction mapping.

A pseudorabies virus recombinant called S-PRV-002 was shown by Southernhybridization to contain a deletion in BamHI #11 fragment and in BamHI#5 fragment (See FIG. 3). This virus is lacking in thymidine kinaseactivity, as shown by a 14C-thymidine incorporation assay described in(15).

Animal Trials Conducted With S-PRV-001 and S-PRV-002

The first set of studies was designed to compare the safety (virulence)and antigenicity (induction of antibody response) of S-PRV-001 andS-PRV-002 with wildtype pseudorabies virus (PRV).

Weaned pigs (4-6 weeks old) that had no prior exposure to PRV wereinoculated with various concentrations of the above 3 viruses. Theanimals were observed daily for 14 days to detect clinical signs of PRVand body temperatures were recorded daily. At designated times, bloodsamples were taken from the pigs and the serum was tested for antibodyto PRV, using a radioimmunodiffusion enzyme-linked immunosorbent assay(RIDEA). At various times post-inoculation, pigs were sacrificed,tissues were removed and assayed for the presence of virus.

The virulence of these three viruses was also evaluated by injectingyoung mice and determining the lethal dose level.

Results of these studies are presented in Tables 1-4.

                  TABLE 1                                                         ______________________________________                                        MOUSE VIRULENCE OF WILD-TYPE,                                                 S-PRV-001 OR S-PRV-002                                                        PSEUDORABIES VIRUSES                                                                                       Mean                                                     Virus                Death                                                    Concen-    No. Dead/ Time  Clinical                                   Virus   tration    No. Inoc. (Hrs.)                                                                              Signs                                      ______________________________________                                        Wild-   10.sup.5   4/4       62    lethargic                                  Type    10.sup.4   5/5       82    severe pruritis                                    10.sup.3   4/5       86    rough coat                                         10.sup.2   0/5       --                                                       10.sup.1   0/5       --                                               S-PRV-001                                                                             10.sup.6   5/5       83    lethargic                                          10.sup.5   5/5       94    slight pruritis                                    10.sup.4   4/5       106   moribund                                           10.sup.3   2/5       153                                                      10.sup.2   0/4       --                                               S-PRV-002                                                                             10.sup.6   0/4       --    none                                               10.sup.5   0/5       --                                                       10.sup.4   0/4       --                                                       10.sup.3   0/4       --                                                       10.sup.2   0/4       --                                               ______________________________________                                         .sup.a Tissue culture infective dose.sub.50 ; TCID.sub.50                

                  TABLE 2                                                         ______________________________________                                        SWINE VIRULENCE OF WILD-TYPE,                                                 S-PRV-001 OR S-PRV-002                                                        PSEUDORABIES VIRUSES                                                                  Virus                                                                         Concen-    Pig    Elevated                                                                             CNS   Other                                  Virus   tration.sup.a                                                                            No.    Temp..sup.b                                                                          Signs.sup.c                                                                         Signs                                  ______________________________________                                        Wild-   10.sup.2   3      +      +     Sneezing                               Type               4      ++     +     --                                             10.sup.4   5      +++    ++    --                                                        6      +      +++   weak,                                                                         depressed                                                 7      ++     +++   weak,                                                                         depressed                                      10.sup.6   8      ++     +++   weak,                                                                         depressed                                                                     died                                                      9      +      ++    --                                                        10     +++    ++    --                                     S-PRV-001                                                                             10.sup.4   13     ++     ++    weak,                                                                         depressed                                                 17     ++     +     sneezing,                                                                     coughing                                                  21     +      --    --                                             10.sup.5   12     +      --    --                                                        14     +      --    --                                                        15     +      --    --                                             10.sup.6   16     +      --    --                                                        18     ++     --    snezzing,                                                                     coughing                                                  20     +      --    --                                     S-PRV-002                                                                             10.sup.2   25     --     --    --                                                        26     --     --    --                                             10.sup.4   27     --     --    --                                                        28     --     --    --                                                        29     --     --    --                                             10.sup.6   30     --     --    --                                                        31     --     --    --                                                        32     --     --    --                                     ______________________________________                                         .sup.a TCID.sub.50                                                            .sup.b elevated rectal temperatures presented as + 105-106°, ++        106-107°, +++ 107-108°.                                         .sup.c CNS signs included + slight ataxia and stiffness of rear legs; ++      severe ataxia of rear legs, slight loss of balance; +++ severe ataxia in      all legs, incoordination, lateral recumency and continued head movements.

                  TABLE 3                                                         ______________________________________                                        SEROLOGIC RESPONSES OF PIGS                                                   INOCULATED WITH WILD-TYPE,                                                    S-PRV-001 or S-PRV-002 PSEUDORABIES VIRUSES                                   Virus                                                                         Concen-        Pig    Antibody Titer.sup.b                                    Virus   tration.sup.a                                                                            No.    Day 14 Day 21 Day 28                                ______________________________________                                        Wild-   10.sup.2   3      32     32     32                                    Type               4      64     >64    >64                                           10.sup.4   5      32     64     32                                                       6      --.sup.c                                                                             --     --                                                       7      64     >64    64                                            10.sup.6   8      --.sup.c                                                                             --     --                                                       9      64     >64    >64                                                      10     >64    >64    >64                                   S-PRV-001                                                                             10.sup.4   13     --.sup.c                                                                             --     --                                                       17     64     >64    >64                                                      21     >64    >64    >64                                           10.sup.5   12     --.sup.c                                                                             --     --                                                       14     >64    >64    >64                                                      15     >64    >64    >64                                           10.sup.6   16     --     --     --                                                       18     >64    >64    >64                                                      20     64     >64    >64                                   S-PRV-002                                                                             10.sup.2   25     8      8      4                                                        26     8      8      4                                             10.sup.4   27     64     NT     32                                                       28     64     32     16                                                       29     32     16     8                                             10.sup.6   30     64     32     16                                                       31     16     16     8                                                        32     32     16     16                                    ______________________________________                                         .sup.a TCID.sub.50                                                            .sup.b RIDEA test; all pigs were <2 on Day 0                                  .sup.c These pigs were sacrificed prior to Day 14                             NT = Not tested                                                          

                                      TABLE 4                                     __________________________________________________________________________    VIRUS ISOLATION FROM PIGS                                                     INOCULATED WITH WILD-TYPE,                                                    S-PRV-001 or S-PRV-002 PSUEDORABIES VIRUSES                                                                     Kidney                                            Virus                       Liver                                             Concen-                                                                           Pig                                                                              Day of   Cere-                                                                             Mid-                                                                              Spinal                                                                            Lung                                        Virus tration.sup.a                                                                     No.                                                                              Sacrifice.sup.b                                                                    Tonsil                                                                            brum                                                                              Brain                                                                             Cord                                                                              Spleen                                      __________________________________________________________________________          10.sup.2                                                                          3  28   --.sup.c                                                                          --  --  --  --                                                    4  28   --  --  --  --  --                                          Wild- 10.sup.4                                                                          5  28   --  --  --  --  --                                          Type      6  12   +   --  --  --  --                                                    7  28   --  --  --  --  --                                                10.sup.6                                                                          8  10   +   +   +   --  --                                                    9  28   --  --  --  --  --                                                    10 28   --  --  --  --  --                                                10.sup.4                                                                          13 12   --  --  --  --  --                                                    17 28   --  --  --  --  --                                                    21 28   --  --  --  --  --                                          S-PRV-001                                                                           10.sup.5                                                                          12 5    +   +   +   --  --                                                    14 28   --  --  --  --  --                                                    15 28   --  --  --  --  --                                                10.sup.6                                                                          16 5    --  --  --  --  --                                                    18 28   --  --  --  --  --                                                    10 28   --  --  --  --  --                                                10.sup.2                                                                          25 28   --  --  --  --  --                                                    26 28   --  --  --  --  --                                          S-PRV-002                                                                           10.sup.4                                                                          27 28   --  --  --  --  --                                                    28 28   --  --  --  --  --                                                    29 28   --  --  --  --  --                                                10.sup.6                                                                          30 28   --  --  --  --  --                                                    31 28   --  --  --  --  --                                                    32 28   --  --  --  --  --                                          __________________________________________________________________________     .sup.a TCID.sub.50                                                            .sup.b Days postinoculation                                                   .sup.c Samples were scored negative after 2 passages in cell culture; all     positive samples occured on first cell culture passage.                  

Wild-type PRV exhibited a mouse LD₁₀₀ of 10³.2 TCID₅₀. The mice werelethargic, had rough coats and pruritis (itching) prior to death. Allpigs inoculated with wild-type PRV had an increase in body temperatureand showed central nervous system manifestations typical of PRVinfection. Virus was recovered from tissues of a pig sacrificed 10 dayspost-inoculation and from another pig sacrificed 12 dayspost-inoculation. By 14 days post-inoculation all of the pigs had PRVantibody which persisted at high levels through the 28-day samplingperiod.

The mouse LD₁₀₀ of S-PRV-001 was 10³.8 TCID₅₀ ; four times more viruswas required to kill all of the mice than was required by wild type. Themean death time of the mice was delayed by 1 to 2 days, indicating thisvirus had lost some of its virulence for mice. Among the 9 pigsinoculated with S-PRV-001 all had an increase in temperature, but only 2of the 9 pigs developed CNS signs and these were less severe thanobserved in pigs inoculated with wild-type virus. Virus was isolatedfrom 1 of 2 pigs sacrificed 5 days post-inoculation. All of these pigshad PRV antibody levels equal to those from wild-type inoculated pigs.

S-PRV-002 was not virulent for mice and caused no clinical signs orfebrile response in pigs. Virus could not be recovered from pigssacrificed at 28 days postinoculation (no pigs were sacrificed earlier).The virus replicated well in the pigs as evidenced by the antibodylevels, which were only slightly lower than measured in the wild-typeand S-PRV-001 inoculated pigs.

The conclusions from these studies are that S-PRV-001 is a very goodantigen but the deletion made was not adequate to provide a safe virusfor use in pigs. On the other hand S-PRV-002 was avirulent in pigs andmice yet still retained its ability to induce a strong antibody responsein pigs.

To test further the utility of S-PRV-002 as a vaccine virus, avaccination/challenge study was conducted in 5-6 week old PRVantibody-free pigs. One group of pigs was reserved as challengecontrols. The vaccinated pigs were observed as described above foradverse reaction following vaccination. At 27 days followingvaccination, the vaccinated and control pigs were challenged byintranasal administration of 10⁴.7 TCID₅₀ of virulent virus. The pigswere observed an additional 14 days for signs of PRV infection.

The results of this study are presented in Table 5.

                                      TABLE 5                                     __________________________________________________________________________    SEROLOGIC AND CLINICAL RESPONSES FOLLOWING                                    VACCINATION WITH S-PRV-002 AND                                                CHALLENGE WITH WILD-TYPE PRV                                                         Post-Vaccination        Post-Challenge                                 Study                                                                             Pig                                                                              Elev..sup.a                                                                       CNS.sup.b                                                                         Antibody.sup.c                                                                        Elev.                                                                             CNS     Antibody                                   Group                                                                             No.                                                                              Temp.                                                                             Signs                                                                             Day 14                                                                            Day 21                                                                            Temp.                                                                             Signs                                                                             Death                                                                             Day 7                                                                             Day 14                                 __________________________________________________________________________        1  --  --  4   8   --  --   +.sup.d                                                                          --  --                                     Vacci-                                                                            2  --  --  32  32  --  --  --  >64 >64                                    nates                                                                             3  --  --  2   2   +   --  --  >64 >64                                        4  --  --  16  8   +   --  --  >64 >64                                        9  --  --  <2  <2  +++ +++ +   --.sup.e                                                                          --                                     Con-                                                                              10 --  --  <2  <2  +++ + ++                                                                              +   --.sup.e                                                                          --                                     trols                                                                             11 --  --  <2  <2  +++ +   --  <2  >64                                        12 --  --  <2  <2  ++  +++ +   --.sup.e                                                                          --                                         13 --  --  <2  <2  ++  --  --  <2  >64                                    __________________________________________________________________________     .sup.a elevated temperature + = 105-106°, ++ = 106-107°, ++     = 107-108                                                                     .sup.b CNS signs included + slight ataxia and stiffness of rear legs; ++      severe ataxia of rear legs, slight loss of balance, +++ severe ataxia in      all legs, incoordination, lateral recumbency and continued head movements     .sup.c RIDEA test                                                             .sup.d Pig died of causes other than PRV                                      .sup.e Pig died prior to day 7                                           

Adverse post-vaccinal reactions, as evidenced by a rise in bodytemperature or demonstration of typical PRV signs, were not seen in thevaccinated animals. All vaccinates developed PRV antibody and thenon-vaccinated controls remained seronegative. Following challenge withvirulent virus, 3 of 5 control pigs had fevers, developed severe CNSsigns and died. One control developed a fever, exhibited transient CNSsigns and recovered. The fifth control developed a fever but did notshow clinical signs of PRV infection. In contrast, 3 of the 4 vaccinatedpigs remained clinically normal, and developed a strong secondaryantibody response. Once vaccinated pig died 4 days post-challengewithout showing clinical signs of PRV. Examination of tissue did notreveal lesions of PRV infection and no virus could be isolated. Thecause of death remains unknown.

The conclusion from this study is that when S-PRV-002 is given at adosage of 10⁴.0 TCID₅₀, it will elicit a strong protective response inthe vaccinated animals. This response is adequate to prevent infectionthat kills 60% of non-vaccinated pigs.

Additional Pseudorabies Virus Constructions

Additional pseudorabies virus constructions with deletions in repeatsequences have been made according to the methods of this disclosure.One construction has a deletion comprised of a 2000bp sequence of DNAfrom the internal repeat and a 1000bp sequence from the adjacent uniquelong region. Results from preliminary tests of this virus in mice andpigs show that it is attenuated. This virus is an example of anattenuated pseudorabies virus with a deletion in a junction region, andis potentially useful as a vaccine for pseudorabies virus disease.

In another example, a pseudorabies virus with a deletion in the repeatregion, specifically between the XbaI site and the HpaI site in BAMHI #5fragment shown in FIG. 2, has been constructed. This virus has not yetbeen tested for virulence.

These examples show that other deletions in repeats can be constructedand indicate that deletions in the repeat sequences attenuate the virus.

References

1. R. W. Price and A. Kahn, Infection and Immunity 34, 571-580, 1981.

2. P. B. Tenser, et al , Journal of General Virology 64, 1369-1373,1983.

3. S. Kit, et al., Ninth International Herpesvirus Workshop, Seattle,August 24-29, 1984.

4. Roizman, et al., Coldspring Harbor Conference on New Approaches toViral Vaccines, September, 1983.

5. R. L Thompson, et al., Virology 131, 180-192, 1983.

6. A. Tanaka, et al., Herpesvirus Meeting, Keystone, Co., 1984.

7. J. M Koomey, et al., Journal of Virology 50, 662-665, 1984.

8. B. Lomniczi, et al., Journal of Virology 49, 970-979, 1984.

9. Ben-Porat, et al., Ninth International Herpesvirus Workshop, Seattle,August 24-19, 1984.

10. Maniatis, et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, 1982.

11. L. E. Post, et al., PNAS 77, 4201-4205, 1980.

12. P. Dierks, et al., Developmental Biology Using Purified Genes.

13. D. D. Brown and C. F. Fox (Eds.) ICN-UCLA Symposium on Molecular andCellular Biology, Acad. Press N.Y., pp. 347-366, 1981.

14. L. E. Post, Mackem, and B. Roizman, Cell 24, 555-565, 1981.

15. Tenser, et al., J. Clinical Microbiology 17, 122-127, 1983.

16. S. Kit, et al., Herpesvirus Meeting, ICN-UCLA Symposium, April 8-13,1984, Abstract #1288.

What is claimed is:
 1. An attenuated pseudorabies virus designatedS-PRV-001 and deposited under ATCC Accession No. VR
 2106. 2. Anattenuated pseudorabies virus designated S-PRV-002 and deposited underATCC Accession No. VR
 2107. 3. A vaccine for pseudorabies virus diseasewhich comprises an effective immunizing amount of the attenuatedpseudorabies virus of claim 1 and a carrier.
 4. A method of immunizingan animal against pseudorabies virus disease which comprisesadministering to an animal an effective immunizing dose of the vaccineof claim
 3. 5. A vaccine for pseudorabies virus disease which comprisesan effective immunizing amount of the attenuated pseudorabies virus ofclaim 2 and a carrier.
 6. A method of immunizing an animal againstpseudorabies virus disease which comprises administering to an animal aneffective immunizing dose of the vaccine of claim
 5. 7. A vaccine ofclaim 3 or 5, wherein the carrier is a physiologically balanced culturemedium containing stabilizing agents.
 8. A vaccine of claim 3 or 5,wherein the effective immunizing amount is from about 10³ to about 10⁹pfu/dose.
 9. A vaccine of claim 8, wherein the effective immunizingamount is from about 10⁴ to about 10⁶ pfu/dose.
 10. A method of claim 4or 6, wherein the animal is a swine, dog, cat, sheep or bovine animal.11. A method of claim 4 or 6, wherein the vaccine is administered byintramuscular, subcutaneous, interperitoneal or intravenous injection.12. A method of claim 4 or 6, wherein the vaccine is administeredintranasally.
 13. A method of claim 4 or 6, wherein the vaccine isadministered orally.